Project Summary The gut microbiota is recognized as a central factor influencing immune responses. This proposal addresses two important questions in understanding how microbes regulate human health: 1) What are the microbe- derived molecules that influence immunity? 2) How does chronic exposure to microbial products alters the phenotype of immune cells in their local environment? Discovering and characterizing the molecules involved in the crosstalk between symbiont microbes and the immune system is a fundamental step towards modulating both the beneficial and harmful potential of the human-microbe relationship. The first major aspect of this project (Aims 1 and 2) uses a series of biochemical approaches to define specific bioactive microbial lipids from human-derived gut microbes that activate invariant natural killer T (iNKT) cells, a specialized lymphocyte subset that recognizes specific lipids as antigens and orchestrates immune responses in multiple contexts. Although animal models have uncovered a role for iNKT cells in immunity, the lipid antigens that they recognize remain unknown in most situations. Here, we study two human symbiont microbes that produce iNKT cell-activating lipids, an uncommon feature among gut anaerobes. To identify the specific molecular lipid species that activate iNKT cells, we will use a newly-developed method, the ?T cell receptor trap,? to capture antigenic lipids. Once purified, these lipids will be characterized by mass spectrometry, including new methods that distinguish sugar stereochemistry, which is of central importance to the activity of lipids that activate iNKT cells. The second major aspect of this project (Aim 3) builds on preliminary transcriptomic data to investigate how chronic exposure to lipid antigens affects the phenotype of iNKT cells in the gut in a mouse model. Since iNKT cells orchestrate immune responses in many situations, and can be either beneficial and harmful, changes to their phenotype in the gut is likely to alter the course of enteral infection, inflammatory bowel disease, colon cancer, or food allergy. To understand how lipids in the gut regulate iNKT cell populations, we will use two reductionist models, feeding mice with experimental diets containing different levels of iNKT cell lipid antigens, or colonization of germ-free mice with lipid antigen-producing microbes. In these models, we will perform high- depth phenotyping of the gut iNKT cell populations. Together, these studies will define lipid antigens from two important members of the human gut microbiota and shed light on the mechanisms by which gut lipid antigens shape iNKT cell populations.